The long term goal of this project is to understand at a fundamental level the response of the polymorphonuclear leukocyte (PMN) to local injury of the body. The PMN detects specific chemical signals that emanate from the site of insult. It crawls toward the signal and, upon reaching the source, it ingests and kills bacteria. Thus, the PMN provides a critical first line of defense against infection and to understand the mechanistic basis for its behavior is of obvious health relavence. Detection of chemical signals by the PMN is accomplished through transduction at the cell membrane of specific receptors coupled to G-proteins. Second messengers activate the motile apparatus to polarize the cell and set it into motion. The polymerization of actin is critical to the response. The present goal is to determine how the chemical signals evoke actin polymerization and how this contributes to the motile response. The first aims are structural: to locate the subcellular sites of actin polymerization following focal stimulation (via a patch pipette) and diffuse stimulation; determine the polarity of the evoked filaments in relation to the membrane. Evoked polymerization is monitored using modified actins whose fluorescent signals increase upon polymerization. Filament polarity and the relation to the membrane will be determined by electron microscopy following labeling with the S-1 fragment of myosin. The next aims are biochemical: indentify the control mechanism for the stimulus evoked actin polymerization. The method is to stimulate intact cells, extract them and probe the extracts for evidence of regulatory activity (including changes in free filament ends, proportion of barbed and pointed free ends, capping and cutting). We shall also determine if the activities so identified depend on known actin binding proteins. The last aims are also biochemical: identify pathways linking transduction to actin polymerization. We shall start by developing an in vitro transduction system using as endpoints the activities characterized in Aim 2.